Enhanced immunogenicity for GPI-anchored antigens

Abstract

Compositions and methods are presented that allow for an enhanced immune response against a GPI-anchored tumor associated antigen by modification of the protein portion of the TAA to include a transmembrane domain and a trafficking signal that directs the modified protein to the endosomal or lysosomal compartment. Most preferably, the modified protein will no longer have a GPI anchor or GPI attachment sequence.

What is claimed is: 
     
       1. A recombinant nucleic acid, comprising:
 a sequence segment encoding a hybrid protein, wherein the hybrid protein comprises an antigen coupled to at least one transmembrane domain and a trafficking element;
 wherein the antigen is a glycophosphatidylinositol (GPI)-anchored protein CEA (carcinoembryonic antigen), and wherein the GPI anchor signal sequence is removed or modified to abolish or render GPI binding less efficient; 
 wherein the transmembrane domain is defined by SEQ ID NO:5; 
 wherein the trafficking element is CD1a (Cluster of Differentiation 1a), CD1c (Cluster of Differentiation 1c), or Lamp1 (Lysosome-associated membrane glycoprotein 1), and directs the recombinant hybrid protein to a sub-cellular location selected from the group consisting of a recycling endosome, a sorting endosome, and a lysosome; and 
 wherein the sequence segment is operably linked to a promoter to drive expression of the hybrid protein. 
 
 
     
     
       2. The recombinant nucleic acid of  claim 1 , wherein the nucleic acid is a viral expression vector or wherein the viral expression vector is an adenoviral expression vector, optionally having a deleted E1 (early region 1) and E2b (early region 2b) gene. 
     
     
       3. The recombinant nucleic acid of  claim 1 , wherein the promoter is a constitutive promoter or an inducible promoter. 
     
     
       4. The recombinant nucleic acid of  claim 1 , wherein the recombinant nucleic acid further comprises a sequence that encodes at least one of a co-stimulatory molecule, an immune stimulatory cytokine, a protein that interferes with or down-regulates checkpoint inhibition, and an adjuvant polypeptide. 
     
     
       5. The recombinant nucleic acid of  claim 4 , wherein the adjuvant polypeptide is calreticulin or HMGB1 (High mobility group box 1). 
     
     
       6. The recombinant nucleic acid of  claim 1 , wherein the transmembrane domain is bound to the C-terminus of the antigen, or wherein the transmembrane domain is coupled to the C-terminus of the antigen via a peptide linker. 
     
     
       7. The recombinant nucleic acid protein of  claim 1 , wherein the trafficking element is bound in-frame to the C-terminus of the transmembrane domain. 
     
     
       8. The recombinant nucleic acid of  claim 1 , wherein the hybrid protein comprises the polypeptide sequence of SEQ ID NO:2. 
     
     
       9. The recombinant nucleic acid of  claim 1 , wherein the hybrid protein comprises the polypeptide sequence of SEQ ID NO:3. 
     
     
       10. The recombinant nucleic acid of  claim 1 , wherein the hybrid protein comprises the polypeptide sequence of SEQ ID NO:4. 
     
     
       11. A recombinant virus comprising the recombinant nucleic acid of  claim 1 . 
     
     
       12. The recombinant virus of  claim 11 , wherein the recombinant virus is a replication deficient virus. 
     
     
       13. A recombinant antigen presenting cell comprising the recombinant nucleic acid of  claim 1 . 
     
     
       14. The recombinant nucleic acid of  claim 3 , wherein the inducible promoter is inducible by hypoxia, IFN-gamma, or IL-8. 
     
     
       15. The recombinant nucleic acid of  claim 4 , wherein the wherein the co-stimulatory molecule is selected from the group consisting of OX40L, 4-1BBL, CD (Cluster of Differentiation)-80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TLIA, ICAM-1, and LFA3. 
     
     
       16. The recombinant nucleic acid of  claim 4 , wherein the immune stimulatory cytokine is selected from the group consisting of IL-2 (Interleukin-2), IL-12 (Interleukin-12), IL-15 (Interleukin-15), IL-15 super agonist (ALT803), IL-21 (Interleukin-21), IPS1 (IFN-β promoter stimulator 1), and LMP1 (Latent membrane protein 1). 
     
     
       17. The recombinant nucleic acid of  claim 4 , wherein the protein that interferes with or down-regulates checkpoint inhibition is an antibody; or an antagonist selected from the group consisting of CTLA-4 (Cytotoxic T-lymphocyte associated protein 4), PD-1 (Programmed cell death protein 1), TIM1 (T-cell immunoglobulin and mucin domain 1) receptor, 2B4, and CD160. 
     
     
       18. The recombinant virus of  claim 11 , wherein the recombinant virus is an adenovirus having a deleted E1 and E2b gene.